Localization and functional difference of MSA-2 paralogs in Babesia bovis merozoites

Babesia bovis merozoite surface antigen 2 (MSA-2) proteins are encoded by members of the msa multigene family and are implicated in erythrocyte recognition and invasion, yet their roles remain only partially understood. We investigated spatiotemporal differences among three MSA-2 paralogs (MSA-2a1, MSA-2a2, and MSA-2b) using epitope-specific rabbit antisera. Alignment of the amino acid sequences of B. bovis T2Bo strain and Texas strain C1 clone showed that MSA-2a1 was highly conserved (99.6% identity) between the parasites, whereas MSA-2a2 (84%) and MSA-2b (94%) were more variable; notably, the least conserved paralog, MSA-2a2, harbored a contiguous 24-amino-acid deletion. Indirect immunofluorescence assays using the antisera revealed apical localization of all three proteins in intraerythrocytic parasites. Following merozoite purification and brief incubation, MSA-2a1 localization was observed on the merozoite surface before attachment, whereas MSA-2a2 and MSA-2b remained at the apical end. Moreover, growth-inhibition assays showed that antiserum Ab-1, which recognizes MSA-2a2 and MSA-2b, inhibited parasite growth by 90.4% after 48 h of incubation, whereas Ab-2, recognizing MSA-2a1 and MSA-2a2 inhibited parasite growth by only 13.1%. These results suggest spatiotemporal differences among MSA-2 paralogs: MSA-2a1 was exposed on the merozoite surface before attachment, whereas MSA-2a2 and MSA-2b retained apical-end at the onset of attachment. Taken together, MSA-2a1, MSA-2a2, and MSA-2b appear to be structurally similar yet functionally distinct proteins that may contribute to the coordinated control of erythrocyte invasion.